In liquid chromatography, a compound is broken down into its components in a chromatographic column, so that these components can be further processed or analyzed by a detector, such as a mass spectrometer. In various liquid chromatography techniques, such as High-Pressure Liquid Chromatography (HPLC), the components of a sample to be separated or analyzed are dissolved in a mobile phase liquid, termed an eluent, and then conveyed by that liquid to a stationary phase within one or more chromatography columns. HPLC analyses are employed in a wide variety of applications, such as drug discovery and development, environmental testing, and diagnostics. In HPLC systems, the chromatographic columns are interconnected to other components by fluidic systems. Such fluidic systems generally include an injection valve, possibly one or more auxiliary valves, various solvent and wash fluid reservoirs, together with various interconnecting fluid tubing lines which are used. Such a fluidic system may be used to supply the liquid, with dissolved sample, to a chromatographic column, and to transfer chromatographically separated dissolved components from the column to, for example, a detector. Selected pressures ranging from substantially atmospheric pressure to pressures on the order of ten thousand pounds per square inch are common in HPLC systems to force the liquid into the column. A detector interfaced to a chromatography system may analyze various samples in serial fashion.
In many situations, there is a need to analyze a large number of samples by HPLC in an efficient manner. To facilitate efficient sample selection and processing, automated analysis systems are available which usually include automatic sample injectors or auto-injectors. Such auto-injectors, often referred to as autosamplers, hold a plurality of samples to be analyzed and are able to feed these in series into a liquid chromatographic analysis system. Typical auto-injectors include a plurality of sample reservoirs, a syringe or syringe-like sample transport system, and automation and computer control systems. The auto-injector system may also include the fluidic system components—the valves, fluid reservoirs, and interconnecting fluid tubing lines—described in the previous paragraph. Auto-injectors commonly mimic cumbersome manual injection methods in which a metered aliquot of a sample is aspirated from a desired sample reservoir into a transfer tubing loop under the action of a syringe pump. The aspiration process is often controlled by pulling back on a plunger or piston to create a negative pressure resulting in aspiration of the sample. Upon reconfiguration of the valve of valves of the fluidic system, the sample may then be transferred to the column. The term “autosampler” is sometimes used to refer to just the aspiration and sample transport portion of such a system.
FIGS. 1A-1B illustrate a known chromatography analysis system including an auto-injector, adapted from that described in United States Patent Application Publication US2009/0145205-A1, in the name of inventor Hochgraeber and incorporated herein by reference. FIG. 1A illustrates the system with the high pressure valve 1 configured in a LOAD position and FIG. 1B illustrates the same system with the valve configured in an INJECT position. The valve has six fluid ports, shown as ports 11, 12, 13, 14, 15 and 16. A high-pressure pump 40 that can supply a constant flow rate of solvent (supplied from a not-illustrated solvent reservoir) under high pressure is connected to port 15. In the switching position of the valve as drawn in FIG. 1A, this flow reaches port 14 through groove 25 of the valve 1, and then reaches a chromatographic column 41. A sample aspiration device, such as sample needle 44, is configured so as to dip into and withdraw a portion of sample from a sample container 43. The sample needle 44 is fluidically connected to port 12 of the valve 1. Instead of being moved into sample container, sample needle 44 can be moved into a waste container 38 to dispose of excess liquid. The needle may be directed so as to access various sample containers 43 or waste containers 38 under the operation of a computer controlled mechanical robot device 39, such as a robotic arm and supporting structures.
Typically, the robot device 39 is operable so as to controllably and automatically position the needle 44 over any of the sample containers 43, waste containers 38, or other containers or ports. The robot device is typically also operable so as to vertically dip the sample needle 44 into a sample container 43 so as withdraw a sample portion and to subsequently lift the sample needle out of the sample container. A syringe or syringe pump 42 for drawing sample liquid is connected to port 11 of valve 1. The two remaining ports 13, 16 are externally connected to one another via a sample loop 50. Sample fluid can thereby be drawn from sample container 43 into sample loop 50 with the aid of syringe or syringe pump 42. The switching position of the valve as drawn is referred to as the LOAD position, since the sample material is being loaded into the sample loop.
In order to feed the sample material into the high-pressure liquid stream, the valve 1 is switched over to a second switching position, which is shown in FIG. 1B. In this configuration, sample loop 50 is looped into the liquid path between pump 40 and column 41. The sample liquid previously drawn into sample loop 50 is thereby transported with the liquid stream coming from pump 40 into column 41, where the chromatographic separation takes place. A detector 45, commonly a mass spectrometer, may be connected downstream of the column. The switching position of the valve as drawn is referred to as the INJECT position, since the sample material is being injected into the high-pressure liquid. The entire process may be repeated for a subsequent sample. Although the sample containers 43 are illustrated, in FIGS. 1A-1B, as glass vials, they could alternatively comprise wells of microtiter plates or any other form of sample container. Sample reservoirs may be sealed with a plastic film or metal foil, or a septum.
FIG. 2 illustrates another known chromatography analysis system including a different commonly-used auto-injector configuration. The system shown in FIG. 2 comprises a separate modular autosampler system 2 that comprises aspiration, transfer and dispense capabilities. The robot system 39 of the autosampler system 2 shown in FIG. 2 is operable so as to aspirate a portion of sample from any one of the sample containers 43 and then dispense the sample portion to a sample injection port 7 of a modular chromatography system 9. The illustration shown in FIG. 2 is schematic; typically, the injection port 7 may be configured so that the sample needle 44 may be inserted into the injection port to as to form a leak-tight seal therebetween during injection of the sample portion. As in the chromatography systems shown in FIG. 1, the system of FIG. 2 comprises a multi-port valve 1. However, the fluid connections of the system of FIG. 2 are plumbed differently from those of the system of FIG. 1 partly because the syringe pump 42 is a component of the autosampler module 2. The valve configuration shown in FIG. 2 is a LOAD configuration in which the sample introduced into injection port 7 is delivered to sample loop 50. With this configuration, any fluid previously in the sample loop 50 is flushed out to waste container 8. In an alternative ELUTE configuration (not illustrated in FIG. 2), the grooves 21, 23, 25 of the valve 1 are re-aligned, by rotation of a rotor of the valve, such that a buffer solution or other solvent is caused to pass through the sample loop 50 under high pressure conditions provided by pump 40, such that the previously loaded sample portion is flushed, together with the buffer or solvent, out of the loop 50 and into column 41.
The auto-injector systems illustrated in FIGS. 1-2 are basic systems which provide limited fluid-routing flexibility and limited ability to change solvents during the course of a separation. More-complex systems are known—comprising one or more additional valves, fluid reservoirs or columns—which provide additional necessary or required functionality. For instance, U.S. Pat. No. 7,588,725, in the names of inventors Ozbal et al. and incorporated herein by reference, teaches an autoinjection system having a sample injection valve, a column control valve and a wash control valve. The sample injection valve has a first position which applies a reduced pressure to a sample sipper tube for aspirating a fluidic sample into the sample sipper tube, and a second position which delivers the fluidic sample to a sample supply loop. The column control valve has a first position which delivers the fluidic sample from the sample supply loop to a sample chromatography column, and a second position which reverses direction of fluid flow through the sample chromatography column to deliver the fluidic sample to a sample analyzer. The wash control valve has a first position which supplies a wash buffer solution to the sample chromatography column in a forward fluid flow direction, and a second position which supplies elution solvent to flush the sample supply loop. Fluidic circuits taught by Ozbal et al. provide the capability of passing a fluid over the insoluble matrix of a chromatography column in a first direction such that an analyte in the fluid binds to the insoluble matrix, and back-eluting an elution fluid over the insoluble matrix in a second direction opposite the first direction to output a sample that includes the analyte. Fluidic circuits taught by Ozbal et al. provide the capabilities of delivering wash buffer solution from the wash control valve to the sample chromatography column; delivering elution solvent from the wash control valve to the sample supply loop and aspirating a second sample fluid while simultaneously outputting a first fluid in the sample loop to an analyzer.
U.S. Pat. No. 6,635,173, in the name of inventor Brann and incorporated herein by reference discloses a multi-column chromatography system which is illustrated herein as FIG. 3 of the present document. As can be seen therein, the system 50 contains an autosampler 51 which includes a plurality of injection valves 54, a plurality of pumps 56, a plurality of columns 58, and a selector valve 60 and a detector 62. Columns 58 may comprise a wide variety of columns useful for chromatographic analysis which can be used to direct a fluid sample into the entrance orifice of a given detector. For example, columns 58 may comprise high performance HPLC columns, capillary electrophoresis columns, gas chromatography columns, flow injection transfer lines, etc. In addition, although not shown, the system may also preferably include a port valve, positioned before the columns, which in the case of a single column system (one or more pumps and one or more columns) operates to load sample in one direction and elute in the opposite direction, as previously described in this document. In the case of a two column system, the port valve provides a similar function, and also provides a loop for eluting solvent.
Each combination of pump 56, injection valve 54, and column 58 (together with any associated tubing, additional valves, additional pumps or additional columns) shown in FIG. 2 may be regarded as a separate chromatographic system operating in parallel with three other such chromatographic systems. In that regard, it can be appreciated that each chromatographic system is controlled by the computer controller 63 to ensure that samples are introduced by the autosampler to avoid overlap at the detector end, and to ensure maximum use of the detector's time as a detector. In that regard, the system 50 may be considered as comprising four (4) independent chromatography systems, wherein each system contains one (1) or more pumps and one (1) or more columns. Each of such independent chromatography systems may comprise two (2) pumps and one (1) column, such that one pump is devoted to loading the column with sample, and one pump for elution.
FIG. 4, reproduced from the aforementioned U.S. Pat. No. 6,635,173, illustrates curves 70, 72, 74, and 76 showing the procedural benefit of controlled staggered/sequenced sample injections as may be performed using the system 50 (FIG. 3). As can be seen, the detector analyzes each curve in sequence. In such regard the detector herein functions to detect and report curve 70, while those samples responsible for curves 72, 74, and 76, although in the process of being eluted in the column, have not yet exited from the column. Such programmed chromatography sequencing is provided herein by a computer control device 63 (FIG. 3) which, upon consideration of when the target sample is likely to exit the column, adjusts the introduction of samples from the autosampler into the columns to sequentially deliver eluant containing sample for sequenced detection. In other words, the computer controller 63 considers the samples in the autosampler, and the input of information concerning their anticipated data-collecting window at the detector, and selects those samples from the autosampler for introduction into the system to maximize detector use.
As indicated in the above discussion, processing of any single sample by a liquid chromatography (LC) system generally begins with an autosampler. Unless otherwise indicated herein, the term “autosampler” is used from this point forward in this document to refer to a modular autosampler, such as the modular autosampler 2 shown in FIG. 2. Unlike operational procedures run by other components, such as pumps and detectors, it is often difficult to determine beforehand the duration of an autosampler procedure. Steps do not necessarily have precise times associated with them. Furthermore, times can vary significantly system to system depending on instrument configuration. In general, such an autosampler executes a programmed procedure of the following general form:                (a) Pre-sample steps (steps executed prior to drawing a sample). This sequence may include wash steps of the syringe and injector, introduction of one or more air gaps (to separate different samples), and movement into position over the position of a sample vial or other sample container. Issues encountered during this phase need not affect sample results. In other words, any malfunctions or other errors encountered during this pre-sample phase will generally not damage or otherwise modify a sample in such a way so as to give incorrect analytical results, since the sample remains in its container. Thus, any such malfunctions or other errors occurring during this phase, if detected by a control system, can possibly be compensated for by simply aborting an existing procedure and re-starting it from the beginning, by switching operations to a backup or concurrently running autosampler or LC system or channel or perhaps, by raising an alarm which will instruct an operator to take corrective action. Spans of time denoting the execution of pre-sample steps are identified by reference symbols including the letter “p” in the accompanying drawings and associated text—for example, a span of time the associated with the execution of pre-sample steps of the jth sample analysis procedure is identified (see FIGS. 5A-5B) with a generalized reference symbol of the form pj. The kth such span of time is identified (see FIGS. 7A-7B) by a generalized reference symbol of the form pj(k).        (b) Sample Transport. Sample transport begins with physically drawing the sample. This step includes physically moving an aspirated sample and may, optionally, include certain sample preparation steps—such as mixing with a reagent, centrifuging, etc. Any issue encountered during this phase may directly affect the sample being transported. Possible problems which may occur during this phase may include leakage of a sample out of a sample needle (for low-viscosity samples), sample evaporation or time-dependent degradation of the sample, either by exposure of the sample to air, to an unfavorable temperature environment or to ambient light. Accordingly, it is desirable to adjust the timing of commencement of this phase so that the sample is held in the needle or other transport device for no longer than is necessary. Spans of time denoting the execution of sample transport steps are identified by reference symbols including the letter “q” in the accompanying drawings and associated text—for example, a span of time associated with the execution of sample transport steps of the jth sample analysis procedure is identified (see FIGS. 5A-5B) with a generalized reference symbol of the form qj. The kth such span of time is identified (see FIGS. 7A-7B) by a generalized reference symbol of the form qj(k).        (c) System Synchronization (“Sync”). The autosampler may wait for confirmation that an LC system is ready to receive an injection. The sync may operate through either software or hardware mechanisms. Because some samples may be reactive or prone to degradation or loss as noted above, it is desirable to minimize such waiting time. These “Ready” signals are identified by reference symbols 81a-81d in FIGS. 5A-5B and reference symbols 85a-85c in FIG. 7A.        (d) Injection. The system injects the sample into the LC system and starts other LC devices, effectively transferring control of the sample. Each injection operation requires a brief but finite time period—these are represented by time periods noted as tinject in FIGS. 5A-5B. The completion of injection is indicated by reference symbols 83a-83d in FIGS. 5A-5B and reference symbols 87a-87b in FIG. 7A. At such times, injection-completed signals may be transmitted from the autosampler the LC channel system.        (e) Post-Injection Operations. The autosampler may execute several other operations on the back end of the program. Spans of time denoting the execution of post-injection steps are identified by reference symbols including the letter “z” in the accompanying drawings and associated text—for example, a span of time associated with the execution of post-injection steps associated with the jth sample analysis procedure is identified (see FIGS. 5A-5B) with a generalized reference symbol of the form zj. The kth such span of time is identified (see FIGS. 7A-7B) by a generalized reference symbol of the form zj(k).        
Full time spans including some or all of the austosampler procedural steps (a)-(e) above are identified by reference symbols including the letter “r” in the accompanying drawings and associated text—for example, time spans r1, r2, r3 and r4 in FIGS. 5A-5B. FIG. 5A illustrates a first example of a conventional timing scheme for coordinating the operation of an autosampler and an LC system. The horizontal axis in FIG. 5A represents time. In FIG. 5A, as well as in FIG. 5B and FIG. 7, boxes at the base of the diagram represent periods of time during which a liquid chromatography channel—such as a liquid chromatography/mass spectrometry (LC/MS) system or a single channel thereof—is busy performing the operations of separating components of and possibly identifying or quantifying chemical species within a previously injected sample. Different patterns applied to boxes at the base of the diagram represent either different samples or different sample analysis procedures, denoted as si (where i is an integer) such as samples or procedures s1, s2 and s3. Boxes drawn in solid lines at the top of the diagrams of FIG. 5 and FIG. 7—specifically, boxes r1, r2, r3 and r4—represent periods of time that a robotic autosampler devotes to performing steps (a)-(e) as listed above; un-patterned boxes drawn in dotted lines represents auto-sampler idle time, either as the result of an intentional delay period or else during which the autosampler is waiting for the LC channel or system to become available.
To improve timing between subsequent samples, a typical strategy, as illustrated in FIG. 5A, has been to start the autosampler as early as possible on subsequent samples. Thus, as may be seen in FIG. 5A, autosampler operational procedure r2, relating to preparation for injection of a sample whose analysis is noted at s2, commences immediately after injection of a prior sample whose analysis is noted at s1. Since the robot operations may require a time period of 1-3 minutes whereas the sample analyses may require substantially longer periods—for instance 4-12 minutes, this so called “Look-Ahead” methodology, may frequently lead to situations in which an autosampler has completed steps prior to injection and thus spends a significant period of time waiting for the “System Sync” to continue. Such waiting periods are denoted by the time intervals Δtr1, Δtr2 and Δtr3 in FIG. 5A. The existence of such waiting periods can have several negative consequences: (i) samples sensitive to temperature or other factors may be affected by the length of time spent in the syringe and also by the potential time variations seen between samples; (ii) samples with very low viscosity may begin to drip or mix across air gaps; and (iii) the probability of losing a sample due to an abort or error of a previous sample is increased.
A second a conventional timing scheme for coordinating the operation of an autosampler and an LC system is illustrated in FIG. 5B. The procedure illustrated in FIG. 5B attempts to overcome the above-noted fact that it is preferable to minimize the time a sample spends in the dispensing syringe or needle by attempting to start the auto-sampler such that it is ready to make an injection—that is, the “pre-injection” steps (a) and (b) are completed—just prior to receipt of a “System Sync” signal. This method utilizes a timing-data database 206 that contains autosampler timing estimates entered manually by users based on record-keeping of the time required to conduct autosampler operations during previous analytical runs. Since the various autosampler operation times may vary between different samples or different sample analysis procedures, such as samples or procedures s1, s2 and s3, different timing estimates should be maintained, in parallel, for each of the various samples or procedures. Also the times required to undertake the various LC sample analyses are expected to vary between the different samples or procedures s1, s2 and s3, etc., as is indicated by the varying widths of the differently patterned boxes along the time axis.
The time estimates in the database 206 (FIG. 5B) are used to calculate predetermined autosampler “lag times” that measure the time between the injection of a sample into the LC system and prior to the commencement of the sequence of autosampler steps associated with the next sample. Thus, for instance, in FIG. 5B, the quantity Δtr2s1 represents the predetermined autosampler lag time that measures the time interval from the commencement of execution of the LC sample or procedure s1 until the beginning of the sequence of autosampler steps, r2, required to execute pre-sample steps (a) and sample transport steps (b) in preparation for injecting the sample associated with the next analysis indicated at s2. Each “lag time”, so defined, is related but to not necessarily identical to a “delay time” which is a wait time inserted between the completion of autosampler post-injection steps and the commencement of the sequence of autosampler steps associated with the next sample. Such delay times, so defined, are indicated by the time segments outlined in dotted lines in FIG. 5B. As indicated in FIG. 5B, the correct calculation of the predetermined delay time or, equivalently, lag time, will cause the autosampler to finish its pre-injection steps (indicated by the shaded boxes p2 and q2) just about at the same time that the LC system transmits the System Sync signal 81b indicating that it is ready to receive the sample associated with the analysis indicated at s2.
Similarly, the quantity Δtr3s2 (FIG. 5B) represents another predetermined autosampler lag time. This lag time measures the time from the commencement of execution of the LC sample or procedure s2 until the commencement of the sequence of autosampler steps, r3, required to prepare to inject the sample associated with the next analysis indicated at s3. In this hypothetical case, FIG. 5B indicates that the lag time and associated delay time (dotted line time segment after post-injection steps z2), as calculated from the user-estimated data, was too short, causing the existence of an un-planned autosampler idle time while the autosampler module waits for receipt of the System Sync signal 81c. The next predetermined autosampler lag time, Δtr4s3 (which in this instance is equivalent to the delay time) is inserted immediately after the commencement of execution of the LC sample or procedure s3 and prior to beginning the sequence of autosampler steps, r4, required to prepare to inject the sample associated with the next analysis indicated at s4. In this particular hypothetical case, FIG. 5B indicates that the lag time, as calculated from the user-estimated data, was too long, thereby causing LC system to idly wait for a period time while the autosampler completes its pre-injection steps after the LC system signaled, at 81d, that it was ready to receive the sample. Such delays in the operation of the LC system can lead to overall analytical inefficiency which can become significant during the running of numerous samples of a batch.
The procedure illustrated in FIG. 5B requires diligence and conscientiousness by users to enter timing settings and to adjust them whenever necessary. Such timing setting adjustments should generally be entered whenever an LC analytical procedure or an autosampler procedure is modified. Typically, these procedures may both be modified in concert. Timing adjustments should also be entered whenever an analytical procedure is transferred to another system, since the timings are generally machine-specific. Failure to make such adjustments can seriously affect multiplexing timing efficiency and, as noted above, can effect overall system efficiency or can cause degradation or loss of samples. Moreover, robotic sample aspiration, transport and dispensing operations and multiplexed chromatography systems (FIGS. 1-3) are typically associated with systems designed for automated screening operations, such as high-throughput screening (HTS) systems involving virtually continuous sequences of analyses and large numbers of samples. Such automated high-throughput systems are often utilized, for instance, in drug-discovery procedures and may be used for clinical applications such as drug-testing or screening for vitamin deficiencies or for diagnostic biomarkers. Such automated screening systems are frequently designed for unattended or overnight operation with little operator intervention other than initial loading of samples. In such systems, sample containers may be accessed in a “random” fashion which is generally unpredictable to a user—as may be the case of the sample access and analysis operations are under the control of a computerized scheduler, as may occur with multiplexed chromatographic systems. Because of these factors, it may be difficult or impossible, in practice, for a user to manually maintain detailed logs of elapsed autosampler times for different sample types or procedures.